Browse
Submit Data
Databases
API
Help

Dataset Information

16 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Optimized two-color super resolution imaging of Drp1 during mitochondrial fission with a slow-switching Dronpa variant.

ABSTRACT: We studied the single-molecule photo-switching properties of Dronpa, a green photo-switchable fluorescent protein and a popular marker for photoactivated localization microscopy. We found the excitation light photoactivates as well as deactivates Dronpa single molecules, hindering temporal separation and limiting super resolution. To resolve this limitation, we have developed a slow-switching Dronpa variant, rsKame, featuring a V157L amino acid substitution proximal to the chromophore. The increased steric hindrance generated by the substitution reduced the excitation light-induced photoactivation from the dark to fluorescent state. To demonstrate applicability, we paired rsKame with PAmCherry1 in a two-color photoactivated localization microscopy imaging method to observe the inner and outer mitochondrial membrane structures and selectively labeled dynamin related protein 1 (Drp1), responsible for membrane scission during mitochondrial fission. We determined the diameter and length of Drp1 helical rings encircling mitochondria during fission and showed that, whereas their lengths along mitochondria were not significantly changed, their diameters decreased significantly. These results suggest support for the twistase model of Drp1 constriction, with potential loss of subunits at the helical ends.

SUBMITTER: Rosenbloom AB

PROVIDER: S-EPMC4246937 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Color-Coded Super-Resolution Small-Molecule Imaging.

Project description:Although the development of super-resolution microscopy dates back to 1994, its applications have been primarily focused on visualizing cellular structures and targets, including proteins, DNA and sugars. We now report on a system that allows both monitoring of the localization of exogenous small molecules in live cells at low resolution and subsequent super-resolution imaging by using stochastic optical reconstruction microscopy (STORM) on fixed cells. This represents a powerful new tool to understand the dynamics of subcellular trafficking associated with the mode and mechanism of action of exogenous small molecules.

| S-EPMC5291120 | biostudies-literature

Demosaiced pixel super-resolution for multiplexed holographic color imaging.

Project description:To synthesize a holographic color image, one can sequentially take three holograms at different wavelengths, e.g., at red (R), green (G) and blue (B) parts of the spectrum, and digitally merge them. To speed up the imaging process by a factor of three, a Bayer color sensor-chip can also be used to demultiplex three wavelengths that simultaneously illuminate the sample and digitally retrieve individual set of holograms using the known transmission spectra of the Bayer color filters. However, because the pixels of different channels (R, G, B) on a Bayer color sensor are not at the same physical location, conventional demosaicing techniques generate color artifacts in holographic imaging using simultaneous multi-wavelength illumination. Here we demonstrate that pixel super-resolution can be merged into the color de-multiplexing process to significantly suppress the artifacts in wavelength-multiplexed holographic color imaging. This new approach, termed Demosaiced Pixel Super-Resolution (D-PSR), generates color images that are similar in performance to sequential illumination at three wavelengths, and therefore improves the speed of holographic color imaging by 3-fold. D-PSR method is broadly applicable to holographic microscopy applications, where high-resolution imaging and multi-wavelength illumination are desired.

| S-EPMC4926095 | biostudies-literature

Dual-color fluorescent nanoparticles showing perfect color-specific photoswitching for bioimaging and super-resolution microscopy.

Project description:Dual-emissive systems showing color-specific photoswitching are promising in bioimaging and super-resolution microscopy. However, their switching efficiency has been limited because a delicate manipulation of all the energy transfer crosstalks in the systems is unfeasible. Here, we report a perfect color-specific photoswitching, which is rationally designed by combining the complete off-to-on fluorescence switching capability of a fluorescent photochromic diarylethene and the frustrated energy transfer to the other fluorescent dye based on the excited-state intramolecular proton transfer (ESIPT) process. Upon alternation of UV and visible light irradiations, the system achieves 100% switching on/off of blue emission from the diarylethene while orange emission from the ESIPT dye is unchanged in the polymer film. By fabricating this system into biocompatible polymer nanoparticles, we demonstrate microscopic imaging of RAW264.7 macrophage cells with reversible blue-color specific fluorescence switching that enables super-resolution imaging with a resolution of 70 nm.

| S-EPMC6626011 | biostudies-literature

Optimized localization analysis for single-molecule tracking and super-resolution microscopy.

Project description:We optimally localized isolated fluorescent beads and molecules imaged as diffraction-limited spots, determined the orientation of molecules and present reliable formulas for the precision of various localization methods. Both theory and experimental data showed that unweighted least-squares fitting of a Gaussian squanders one-third of the available information, a popular formula for its precision exaggerates beyond Fisher's information limit, and weighted least-squares may do worse, whereas maximum-likelihood fitting is practically optimal.

| S-EPMC3127582 | biostudies-literature

Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy.

Project description:Single Molecule Localization super-resolution Microscopy (SMLM) has become a powerful tool to study cellular architecture at the nanometer scale. In SMLM, single fluorophore labels are made to repeatedly switch on and off ("blink"), and their exact locations are determined by mathematically finding the centers of individual blinks. The image quality obtainable by SMLM critically depends on efficacy of blinking (brightness, fraction of molecules in the on-state) and on preparation longevity and labeling density. Recent work has identified several combinations of bright dyes and imaging buffers that work well together. Unfortunately, different dyes blink optimally in different imaging buffers, and acquisition of good quality 2- and 3-color images has therefore remained challenging. In this study we describe a new imaging buffer, OxEA, that supports 3-color imaging of the popular Alexa dyes. We also describe incremental improvements in preparation technique that significantly decrease lateral- and axial drift, as well as increase preparation longevity. We show that these improvements allow us to collect very large series of images from the same cell, enabling image stitching, extended 3D imaging as well as multi-color recording.

| S-EPMC4938622 | biostudies-literature

124-Color Super-resolution Imaging by Engineering DNA-PAINT Blinking Kinetics.

Project description:Optical super-resolution techniques reach unprecedented spatial resolution down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. Here, we introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells, and finally experimentally demonstrate 124-plex super-resolution imaging within minutes.

| S-EPMC6463241 | biostudies-literature

Large plasmonic color metasurfaces fabricated by super resolution deep UV lithography.

Project description:In this paper, we demonstrate plasmonic color metasurfaces as large as ∼60 cm2 fabricated by deep UV projection lithography employing an innovative combination of resolution enhancement techniques. Briefly, in addition to the established off-axis dipole illumination, double- and cross-exposure resolution enhancement of lithography, we introduce a novel element, the inclusion of transparent assist features to the mask layout. With this approach, we demonstrate the fabrication of relief arrays having critical dimensions such as 159 nm nanopillars or 210 nm nanoholes with 300 nm pitches, which is near the theoretical resolution limit expressed by the Rayleigh criterion for the 248 nm lithography tool used in this work. The type of surface structure, i.e. nanopillar or nanohole, and their diameters can be tailored simply by changing the width of the assist features included in the mask layout. By coating the obtained nanopatterns with thin layers of either Au or Al, we observe color spectra originating from the phenomenon known as localized surface plasmon resonance (LSPR). We demonstrate the generation of color palettes representing a broad spectral range of colors, and we employ finite element modelling to corroborate the measured LSPR fingerprint spectra. Most importantly, the ∼60 cm2 nanostructure arrays can be written in only a few minutes, which is a tremendous improvement compared to the more established techniques employed for fabricating similar structures.

| S-EPMC9419299 | biostudies-literature

The LRRK2 Variant E193K Prevents Mitochondrial Fission Upon MPP+ Treatment by Altering LRRK2 Binding to DRP1.

Project description:Mutations in leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including 13 putative armadillo-type repeats at the N-terminus. In this study, we analyzed the functional and molecular consequences of a novel variant, E193K, identified in an Italian family. E193K substitution does not influence LRRK2 kinase activity. Instead it affects LRRK2 biochemical properties, such as phosphorylation at Ser935 and affinity for 14-3-3?. Primary fibroblasts obtained from an E193K carrier demonstrated increased cellular toxicity and abnormal mitochondrial fission upon 1-methyl-4-phenylpyridinium treatment. We found that E193K alters LRRK2 binding to DRP1, a crucial mediator of mitochondrial fission. Our data support a role for LRRK2 as a scaffolding protein influencing mitochondrial fission.

| S-EPMC5835904 | biostudies-literature

Multi-color live-cell super-resolution volume imaging with multi-angle interference microscopy.

Project description:Imaging and tracking of near-surface three-dimensional volumetric nanoscale dynamic processes of live cells remains a challenging problem. In this paper, we propose a multi-color live-cell near-surface-volume super-resolution microscopy method that combines total internal reflection fluorescence structured illumination microscopy with multi-angle evanescent light illumination. We demonstrate that our approach of multi-angle interference microscopy is perfectly adapted to studying subcellular dynamics of mitochondria and microtubule architectures during cell migration.

| S-EPMC6240104 | biostudies-literature

Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection.

Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus within 20 years that gave rise to a life-threatening disease and the first to reach pandemic spread. To make therapeutic headway against current and future coronaviruses, the biology of coronavirus RNA during infection must be precisely understood. Here, we present a robust and generalizable framework combining high-throughput confocal and super-resolution microscopy imaging to study coronavirus infection at the nanoscale. Employing the model human coronavirus HCoV-229E, we specifically labeled coronavirus genomic RNA (gRNA) and double-stranded RNA (dsRNA) via multicolor RNA-immunoFISH and visualized their localization patterns within the cell. The exquisite resolution of our approach uncovers a striking spatial organization of gRNA and dsRNA into three distinct structures and enables quantitative characterization of the status of the infection after antiviral drug treatment. Our approach provides a comprehensive framework that supports investigations of coronavirus fundamental biology and therapeutic effects.

| S-EPMC8202426 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data